Assay for anti-viral agent

ABSTRACT

There is provided an assay for selecting an anti-viral agent effective against picornavirus infection. The assay selects an agent which disrupts the association between picornavirus protein 2C and the host cell protein D-AKAP2, which association has been found to be essential for successful viral replication. Also disclosed are anti-viral agents having such properties and a method of combating picornavirus replication.

The present invention relates to an assay for an anti-viral agent forpicornaviruses.

BACKGROUND OF THE INVENTION

Picornaviruses are single positive strand RNA viruses. The genome ofapproximately 7,500–8,250 bases contains a single long open readingframe encoding a large polyprotein, usually approximately 230 kDa. Thepolyprotein is processed auto-proteolytically by virus-encodedproteases. The ‘primary’ cleavages separate the polyprotein into thestructural capsid protein (P1), and non-structural proteins involved invirus replication (P2 and P3), see FIG. 1. All three primary proteins(P1, P2 and P3) undergo post-translational cleavage to produce matureproteins (reviewed in reference [1]).

In the case of the rhino- and enteroviruses this processing is achievedby the 2A proteinase cleaving at its own N-terminus, whilst in theaphtho- and cardioviruses the 2A protein mediates cleavage at its ownC-terminus^([1]). In the latter viruses this cleavage is mediated by anoligopeptidic tract (18 amino acids) corresponding to the complete 2Aprotein of Foot-and-Mouth Disease Virus (FMDV), or, just the C-terminalregion of the larger (˜145 amino acids) cardiovirus 2A protein^([2)].The sequence is able to mediate cleavage in entirely foreign proteincontexts^([3]) and has been used in a wide range of co-expressionstudies^([4-6]). The corresponding primary cleavage in the hepato- andparechoviruses is mediated by the 3C proteinase (see FIG. 1).

It is known that large numbers of membrane vesicles are produced in thecytoplasm of picornavirus-infected cells^([7]). These vesicles arethought to arise from the transitional smooth areas of the EndoplasmicReticulum (ER) that produce transport vesicles which move proteins fromthe ER to the Golgi^([8]). It is also known that Brefeldin-A, a drugthat blocks the formation of ER-derived transport vesicles, is one ofthe most potent known inhibitors of picornavirus replication^([9,10])and that normal membrane transport from the ER to the Golgi is blockedin picornavirus infected cells^([11,12]). Picornavirus infectionappears, therefore to change the balance of membrane budding and fusionwhich is normally maintained between the ER and Golgi compartments.Transport vesicles leaving the ER do not appear to fuse with the Golgi,but are diverted from the secretory pathway to function at sites ofvirus replication, explaining why the Golgi is absent later ininfection. Indeed, the loss of the Golgi is coincident with theformation of virus replication complexes in the cytoplasm. These appearas rosettes of large vesicles surrounding a compact central membranesystem.

As early as 1982 Semler and co-workers demonstrated the association ofthe non-structural protein 3AB with membranes^([13]). Othernon-structural viral proteins 3AB, 2BC, 2B, 2C have since been shown tolocalise to crude membrane fractions^([14]), and to the ER^([15]). Themembrane association of 3A and 3AB has been explained by the presence of20 hydrophobic amino acids at the C-terminus of 3A, and deletion of thisregion abrogates membrane binding. The membrane association of 2C and2BC was proposed via an amphipathic helix that produces a hydrophobicpatch at the N-terminus of 2C^([12]).

Importantly, protein 2C has been localised to electron-dense coatswithin the regions of ER membrane producing cytoplasmic vesicles, andwithin membrane vesicles of the replication complex^([14]). Theobservation that 3A, 3AB, 2BC and 2B bind membranes makes them strongcandidates as inhibitors of membrane traffic in cells. Indeed recentstudies show that 2BC and 2C induce the formation of vesicles^([14]),and 3A and 2B block secretion, when expressed alone in cells^([11, 12]).

In picornaviruses, RNA replication is solely a function of the P2 and P3regions of the polyprotein. A full-length negative sense copy of thegenomic, positive sense, RNA is synthesised and acts as a template forthe synthesis of full length positive RNA strands. In the case of theentero- and rhinoviruses 2A is a proteinase^([1]) and forms part of theP2 primary cleavage product. The function of protein 2B is unknown,protein 2C is discussed in detail below, protein 3A is thought tofunction as a ‘donor’ for the protein 3B (or VPg) which becomescovalently bound to the 5′ terminus of both positive and negative strandRNA. Protein 3C is a proteinase (see reference [1]), which serves tomediate a primary cleavage between P2 and P3 and subsequently processesP2 and P3 (‘secondary’ cleavages).

Protein 2C is the second largest mature protein (approximately 37 kDa)and is the most highly conserved picornavirus protein. Indeed,homologues of 2C are found within other members of the picornavirus‘supergroup’—comprising both (non-picornavirus) animal viruses(caliciviruses, hepatitis E viruses; various insect viruses) andnumerous plant viruses (comoviruses, nepoviruses).

Based on sequence alignments it was proposed that protein 2C containedNTP binding motifs^([16]) and could function as a helicase. Although ithas not proved possible to demonstrate helicase activity this notion hasbeen stated in the literature and is believed to be correct within thecurrent understanding of picornavirus protein function^([41-44]).

Protein 2C is known to have ATPase/GTPase activity^([17,18]). Membranebinding activity maps to the N-terminal region (amino acids 21–54) ofpoliovirus 2C, containing one of the putative amphipathic helices (SeeFIG. 2, panel A). Protein 2C is not glycoslyated nor cleaved by signalpeptidases^([19]). Expression of 2C induces membrane proliferation andblocks the exocytic pathway^([20,21]).

Furthermore, 2C is an RNA-binding protein with sites mapped to the N-and C-terminal regions (amino acids 21–45 and 312–319)^([19]),summarised in FIG. 2, panel A. The RNA sequences bound by 2C weredetermined to be a stem of a clover-leaf structure SEQ ID NO:7 (see FIG.2, panel B) initially characterised in the positive RNA sense andpredicted to be present in the negative sense (anti-genome) RNA.Interestingly, one can see that one component of the RNA-binding domainis contained within that determined for membrane binding.

The specificity of signalling molecules is greatly enhanced by theirsub-cellular compartmentalisation within the cell. The distribution ofthese molecules is governed by their association with (membrane-bound)anchoring proteins which place these enzymes close to their appropriateeffectors (reviewed in [20–22]). The dual-specificity kinase anchoringprotein 2 (D-AKAP2) binds the regulatory subunits (both RI and RII) ofprotein kinase A (PKA). Originally identified by their co-purificationwith RII after cAMP-sepharose chromatography^([23]), AKAPs were furthercharacterised using their property of binding RII immobilised tonitrocellulose or similar solid-phase supports^([24]). D-AKAP2 sequencesare available for mouse^([25]), human^([26]) and we have completelysequenced the porcine D-AKAP2^([27]) identified by the yeast screen. Ineach case a putative ‘regulator of G-protein signalling’ (RGS) domain ispresent, although sequence similarities between D-AKAP2 and the RGSdomains of other proteins is low. The binding of PKA regulatory subunitsis thought to be mediated by an amphipathic helix^([21]). Inspection ofaligned D-AKAP2 sequences shows similarity between the C-terminus andRII binding sites determined for other AKAPs^([21]). The PKA(RII)/D-AKAP2 binding is high (low nanomolar) affinity^([28]) but doesrequire prior dimerisation of the RII subunit^([19]). D-AKAP2 shows awide tissue distribution although its location(s) at the sub-cellularlevel has not been determined.

It has now be found that a strong interaction exists between FMDVprotein 2C and the cellular membrane protein D-AKAP2. When FMDV 2C isused as ‘bait’ in the 2-hybrid yeast screen, a strong interaction isobserved with the C-terminal region (50 amino acids) of D-AKAP2. Theresults from our genetic screen have been confirmed by directbiochemical assays. D-AKAP2 binds protein kinase A (PKA) and has dualspecificity for both the regulatory subunits (RI and RII) of this enzyme(see above). The yeast two-hybrid 2A:D-AKAP2 interaction was confirmedby direct biochemical binding assays using FMDV 2C translated in vitroand from infected-cell lysates. We extended these observations by theanalysis of a site-directed mutagenetic form of 2C. A single mutation inthe NTP-binding motif (K¹¹⁶ to Q) renders this form of 2C unable toreorganise the membrane structure of transfected cells but does notalter the binding to D-AKAP2 in biochemical assays.

Since the membrane binding region of 2C has been mapped to residues 21to 54, we assume this is the site of interaction with D-AKAP2.

Whilst the present invention is founded upon observations made with FMDV2C, the strong homology between the 2C proteins of all picornavirus,caliciviruses, comoviruses, nepoviruses etc. is recognised in the artand acknowledged in the literature.

The reference to “picornavirus protein 2C” herein refers not only to the2C protein of picornaviruses alone, but also to the 2C protein of thepicornavirus “supergroup” which includes calciviruses, hepatitis Eviruses, comoviruses and nepoviruses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the polyprotein expressed from theRNA genome of different picomaviruses, and shows the capsid (P1), P2 andP3 regions.

FIG. 2A is a schematic illustration of the domains in protein 2C ofpoliovirus and shows the putative amphipathic helices, membrane binding,RNA binding and NTP binding domains.

FIG. 2B shows a picomavirus RNA sequence (SEQ ID NO: 7) in a secondary(clover leaf) structure, in which the “stem” forms the binding site forprotein 2C.

STATEMENT OF THE INVENTION

The present invention provides an assay to select agents which disruptthe interaction of picornavirus protein 2C with D-AKAP2.

Preferably the assay selects agents which bind specifically to the 2Cprotein or to D-AKAP2. Generally the assay looks for agents whichdisrupt the 2C:D-AKAP2 association to the extent that replication of thepicornavirus is hindered or prevented. Optionally the assay is conductedin an in-vitro system.

The present invention provides an anti-viral agent to combat (ie toprevent or hinder) replication of a picornavirus, said agent disruptingthe association between picornavirus protein 2C and D-AKAP2 which occursduring viral replication in a host cell.

The anti-viral agent may be a peptide (for example, a synthetic peptide)or more preferably may be a non-peptidal compound having peptidomimeticproperties, ie. is able to mimic a peptide functionally. Such anon-peptidal compound or certain synthetic peptides will be preferred ifresistant to enzymic breakdown by peptidases. An example of a syntheticpeptide resistant to peptidases includes retro-inverso (RI) peptides,that is peptides formed using the D-isomer of naturally occurringL-amino acids. Suitable anti-viral compounds may include peptides havingan amino acid sequence derived from the N-terminal 20 amino acids of 2C,a functional equivalent of such a peptide, or a peptidomimetic compoundtherefor. The anti-viral agent desirably binds specifically to the 2Cprotein or to D-AKAP2.

The anti-viral agent is preferably effective against a picornavirusselected from polio, FMDV, enteroviruses, rhinoviruses, hepatitis Aviruses, parechoviruses, aphthoviruses, cardioviruses and the like.

In a further aspect, the present invention provides an assay todetermine the ability of a test substance to interfere with (preferablyto inhibit) the association of picornavirus protein 2C with D-AKAP2,said assay comprising:

-   -   (i) providing a first component consisting of picornavirus        protein 2C;    -   (ii) providing a second component consisting of D-AKAP2;    -   (iii) exposing said first component to a test substance followed        by addition of said second component, or exposing said second        component to said test substance followed by addition of said        first component, or exposing said first component to said second        component followed by addition of said test substance;    -   (iv) incubating the mixture obtained in step (iii) for a time        period and under conditions suitable to allow interaction        between the components and/or the test substance;    -   (v) removal of any unassociated first or second component and/or        test substance;    -   (vi) detecting the presence, and optionally determining the        amount, of associated first and second components.

The first or second components may be localised on a surface, such as ablotting membrane or an assay plate for ELISA etc.

Detection of the presence and/or amount of second component associatedwith the first component may be conducted by any convenient means.Generally detection may be via an antibody, for example monoclonalantibody, the presence of which is established by exposure to a secondlabelled monoclonal antibody in a typical ELISA-style assay.Alternatively, one of the first and second components may be labelled(eg radioactively, fluorescently or enzymically) to determine itsbinding to the other component.

In a yet further aspect, the present invention provides a method ofcombating (especially preventing or hindering) replication of apicornavirus, said method comprising providing an anti-viral agentcapable of disrupting the association between picornavirus protein 2Cand D-AKAP2 which occurs during viral replication in the host cell.

Generally said agent is able to bind specifically to 2C or to D-AKAP2thereby inhibiting the interaction between these two components whichoccurs during normal picornavirus replication.

The assay described above can also be used to study cellular biologicalaspects of the viral infection in cells or to study and elucidate thestructure of the D-AKAP2:2C complex. Elucidation of these structureswould provide a quantum leap in the understanding of virus replicationand provide new potential drug targets.

Optionally the assay may utilise a co-expression construct able toco-express D-AKAP2 together with the virus replication proteins whichwill self-process and associate from a [P2P3] polyprotein. The constructtakes advantage to the fact that the viral proteins are expressed as aself-processing polyprotein. Advantageously D-AKAP2 can be linked to theP2/P3 region via an appropriate 2A proteinase cleavage site. In thismanner co-expression of all the components of a replication complextogether with D-AKAP2 is achieved.

An example of an assay according to the invention is describedhereinbelow:

EXAMPLE

(i) Generation of Reagents

We have already cloned and expressed the C-terminal region of porcineD-AKAP2 in E. coli as a glutathione S-transferase (GST) fusion protein.We have cloned porcine D-AKAP2 and have access to human D-AKAP2 cDNA.These will be expressed and used to raise (rabbit) mono-specificantibodies. We have cloned and expressed FMDV 2C in E. coli as aglutathione S-transferase (GST) fusion protein^([31]). Differentiatinginfection from vaccination in foot-and-mouth disease using a panel ofrecombinant, non-structural proteins in ELISA. Vaccine 16. 446–459) andused this expressed material to raise (rabbit) antibodies. The titreswere low, however, and this work needs to be (partially) repeated togenerate higher affinity/titre antibodies. We have also cloned andexpressed virus replication proteins 3A, 3B, 3C and 3D^([31, 37, 38]).

(i) FMDV 2C—E. coli Expression

FMDV 2C protein sequences were cloned into the pGEX E. coli expressionvector (Pharmacia) as described (M. Flint, Thesis).

(ii) FMDV 2C—Transcription In Vitro

Sequences encoding 2C were amplified using the polymerase chain reaction(PCR) from plasmid pMR15^([45]). Unique BamHI and EcoRI sites werecreated at the termini and the doubly restricted, gel purified, PCRproduct was ligated into the vector pcDNA3.1 (Invitrogen), similarlyrestricted.

(iii) GST:D-AKAP2 Fusion Protein

The D-AKAP2 specific clone IAP3 (identified by the yeast 2-hybridscreen) was used as the template in the PCR. D-AKAP2 sequences wereamplified using oligonucleotide primers;

-   AKAPF (5′-AAAGGATCCAAAGGGTCCATGTTCTCACAAGC-3′) SEQ ID NO:1 and-   AKAPR (5′-AAAGAATTCTCACAGCTTGGCAGAGGTCTCC-3′) SEQ ID NO: 2.

The PCR product was doubly restricted with BamHI and EcoRI, gelpurified, and ligated into the vector pGEX-2T (Pharmacia) similarlyrestricted. The GST:D-AKAP2 fusion protein (GST-IAP3) was expressed inE. coli strain BL21(DE3). Overnight cultures were grown at 37° C., usedto inoculate fresh Luria broth, and the culture grown at 37° C., withvigourous aeration, until the optical density of the culture was 0.6.Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was then added to a finalconcentration of 1 mM and the culture incubated for a further 1 hr (37°C., aeration). Cells were harvested by centrifugation and the pelletsresuspended in ice-cold phosphate buffered saline (PBS). Cells weredisrupted by two rounds of sonication (10 s each, cooling on ice betweenbursts). The mixture was clarified by centrifugation (13,000 g, 2 min,4° C.) the supermatant then being mixed with a slurry of glutathionesepharose beads for 10 mins at room temperature. The beads were thenwashed 3 times with PBS prior to use in the 2C binding experiments.

(iv) Mutated Forms of FMDV 2C

Using the overlap PCR technique the NTPase motif “A” of 2C was mutatedby a single coding change (Lys116->Gln). PCR Reactions were performedusing plasmid pMR15 as the template and oligonucleotide primers.

5′2C (5′GATATCGGATCCATGCTCAAAGCACGTGACATC-3′) (SEQ ID NO:3) and 3′2C(Lys116Gln) (5′-GTTTGCAAGGAAGCTGCCGCCCTGGCCCTGGCCAGATTT-3′) (SEQ IDNO:4)and a second reaction using primers;

5′2 (Lys116Gln) (5′-AAATCTGGCCAGGGCGGCAGCTTCCTTGCAAAC-3′) (SEQ ID NO:5)and 3,2c (5′GATATCGAATTCTCACTGCTTGAAGATCGGGTG-3′) (SEQ ID NO:6).

The PCR products from each reaction were purified, added, then furtheramplified using primers 5′2C and 3′2C. The product from this overlap PCRreaction was restricted with BamH1 and EcoR1 and ligated into pGEX-2T,similarly restricted.

(v) FMDV 2C:GST-IAP3 Binding Assay

Transcription vector cDNA clones encoding the wild-type FMDV 2C proteinor the mutated form of 2C (Lys116->Gln) were translated in vitro usingthe TnT Quick rabbit reticulocyte lysate system (Promega), as per themanufacturer's instructions. Proteins were radio-labelled using³⁵S-methinone. In vitro translation mixtures were incubated with eitherGST protein bound to glutathione sepharose beads, or the GST-IAP3 fusionprotein bound to glutathione sepharose beads. Typically, 10 μl oftranslation mixture was incubated with 10 μl of the bead slurry (50%suspension in PBS) with bound GST or GST-IAP3 in 1 ml binding buffer.Binding assays were performed at 4° C. for 30 minutes, the mixturecentrifuged to separate the unbound fraction from the beads. Beads werethen washed four times in binding buffer prior to analysis by SDS PAGE.

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1. An assay to determine the ability of a test substance to interferewith the association of protein 2C of a picornavirus or a protein 2Chomolog from a calicivirus with dual-specificity kinase anchoringprotein 2 (D-AKAP2), said assay comprising: i) providing a firstcomponent consisting of protein 2C of a picornavirus or a protein 2Chomolog from a calicivirus; ii) providing a second component consistingof DAKAP2; iii) exposing said first component to a test substancefollowed by addition of said second component, or exposing said secondcomponent to said test substance followed by addition of said firstcomponent, or exposing said first component to said second componentfollowed by said test substance; iv) incubating the mixture obtained instep (iii) for a time period and under conditions suitable to allowinteraction between the components and/or the test substance; v) removalof any unassociated first or second component and/or test substance; andvi) detecting the presence, and optionally determining the amount, ofassociated first and second components.
 2. The assay as claimed in claim1 which is conducted in vitro.
 3. The assay as claimed in claim 1wherein either the first or second components is localised on a surface.4. The assay as claimed in claim 1 wherein association of said first andsecond components is detected by using an antibody, a radio-label,fluorescence, or an enzymic-label.
 5. The assay as claimed in claim 1wherein picornavirus protein 2C and D-AKAP are expressed from aco-expression construct.
 6. The assay as claimed in claim 5 wherein saidco-expression construct expresses D-AKAP2 and also expressespicornavirus protein 2C, wherein said protein 2C is present as part of aP2/ P3 polyprotein having a 2A proteinase cleavage site, and whereinsaid DAKAP2 is linked to the P2/ P3 polyprotein via a 2A proteinasecleavage site.